JARQ 49 (4), 351 - 362 (2015) http://www.jircas.affrc.go.jp Anthracnose Fungi with Curved Conidia, Colletotrichum spp. belonging to Ribosomal Groups 9-13, and Their Host Ranges in Japan Toyozo SATO1*, Jouji MORIWAKI2 and Shigeru KANEKO1 1 Genetic Resources Center, National Institute of Agrobiological Sciences (Tsukuba, Ibaraki 305-8602, Japan) 2 Horticulture Research Division, NARO Kyushu Okinawa Agricultural Research Center, National Agriculture and Food Research Organization (Kurume, Fukuoka 839-8503, Japan) Abstract Ninety fungal strains with falcate conidia belonging to Colletotrichum spp. classified into the ribosomal groups 9-13 (the RG 9-13 spp.) and preserved at the NIAS Genebank, Japan were re-identified based on molecular phylogenetic analysis of the internal transcribed spacer (ITS) region of the rRNA gene, sequences of the glyceraldehyde 3-phosphate dehydrogenase, chitin synthase 1, histone3, and actin genes, and partial sequences of β-tubulin-2 (TUB2) genes, or by BLASTN searches with TUB2 gene sequences. Seventy strains were reclassified into nine recently revised species, C. chlorophyti, C. circinans, C. dematium sensu stricto, C. lineola, C. liriopes, C. spaethianum, C. tofieldiae, C. trichel- lum and C. truncatum, whereas 20 strains were grouped into four unidentified species. RG 9, 10 and 12 corresponded to the C. spaethianum, C. dematium and C. truncatum species complex, respectively, while RG 11 and 13 agreed with C. chlorophyti and C. trichellum, respectively. Phylograms derived from a six-locus analysis and from TUB2 single-locus analysis were very similar to one another with the exception of the association between C. dematium s. str. and C. lineola. Thus, TUB2 partial gene sequences are proposed as an effective genetic marker to differentiate species of RG 9-13 in Japan except for C. dematium s. str. and C. lineola. Thirty-two plant species were identified as new hosts for seven of the species found in this study except for C. circinans and C. trichellum; and two unidentified species. Colletotrichum chlorophyti, C. lineola, C. liriopes, C. spaethianum, and C. truncatum were regarded as polyphagous, whereas C. trichellum and Colletotrichum sp. (Ra), designated tentatively in this study, appeared to have specific pathogenicity to single hosts, Hedera rhombea and Raphanus sati- vus var. hortensis, respectively. Conidial curvature properties, "outer curvature," "inner curvature" and "height/width ratio" successfully represented conidial shape parameters. Conidial curvature properties of the species in RG 9, 10 and 12 were found to correlate with the species complexes. Discipline: Agricultural Environment Additional key words: β-tubulin-2, conidial curvature properties, molecular phylogenetic analyses, re-identification, species complex Introduction with Colletotrichum graminicola (Ces.) G.W. Wilson and Colletotrichum dematium (Pers.) Grove in addition to its The genus Vermicularia Tode (1790) was established three forma based on morphology, C. graminicola and C. for anthracnose fungi with falcate conidia and setae in dematium have been regarded as representatives of patho- acervuli. More than 280 species have been described with gens on the Poales grasses and other plants, respectively nearly every host plant identified (Anonymous 2014). More (Arx 1981, 1987, Sutton 1980, 1992). More than 230 strains species with falcate conidia were also described as species of Colletotrichum in Japan, including those with falcate in Colletotrichum Corda (1831). Since Arx (1957) synony- conidia and setae, were classified into 20 ribosomal groups mized many Vermicularia as well as Colletotrichum species (RG) based on molecular phylogenetic analyses of the This paper reports results obtained in the NIAS Genebank project sponsored by the Ministry of Agriculture, Forestry and Fisheries, Japan. *Corresponding author: e-mail [email protected] Received 24 October, accepted 3 April 2015. 351 T. Sato et al. internal transcribed spacer (ITS) region of the rRNA gene the β-tubulin-2 (TUB2) genes. Genomic DNA extracted (Moriwaki et al. 2002). This analysis showed that six and according to the procedure described by Moriwaki et al. three species were closely related to C. graminicola and C. (2002) was used as a template for the following polymerase dematium, respectively. The latter three species, Colletotri- chain reaction (PCR) analyses. ITS, GAPDH, CHS-1, chum circinans (Berk.) Voglino, Colletotrichum truncatum HIS3, ACT and TUB2 genes were amplified and sequenced (Schwein.) Andrus & W. D. Moore, and Colletotrichum using the primer pairs ITS5 & ITS4 (White et al. 1990), trichellum (Fr.) Duke, were placed on blanches of RG 10, GDF1 & GDR1 (Guerber et al. 2003), CHS-354R & 11 and 13 in the phylogenetic tree, respectively, while CHS-79F (Carbone & Kohn 1999), CYLH3F & CYLH3R strains of C. dematium were classified into both RG 9 and (Crous et al. 2004), ACT-512F & ACT-783R (Carbone & 12. These species belonging to ribosomal groups 9-13 have Kohn 1999) and T1 (O’Donnell & Cigelnik 1997) & Bt2b been tentatively designated as “the RG 9-13 spp.” in this (Glass & Donaldson 1995), respectively. Each gene region study. After Moriwaki et al. (2002), strains pathogenic was amplified with Taq polymerase (TaKaRa, Otsu, Japan) to the Poales grasses and that are related to C. gramini- in a GeneAmp 9700 (Applied Biosystems, Foster City, CA, cola were re-classified into 14 species based on a molecular USA). Cycling conditions for amplification of ITS were phylogenetic analysis with the ITS, the DNA lyase gene 94°C for 3 min, followed by 35 cycles of 94°C for 30 sec- (Apn2), the mating type Mat1-2 (Mat1/Apn2) gene and 55°C for 1 min - 72°C for 1 min, and a final step at 72°C for the manganese superoxide dismutase gene (Sod2) (Crouch 5 min. Conditions for the remaining five genes were 94°C et al. 2009). Conversely, strains pathogenic to other plants for 5 min, followed by 40 cycles of 94°C for 30 sec- 52°C and related to C. dematium were examined based on a for 30 sec- 72°C for 30 sec, and a final step at 72°C for molecular phylogenetic analysis with six genes/regions as 7 min. PCR products were purified using a QIAquick PCR described below and re-classified into 20 species, including Purification kit (Qiagen, Chatsworth, CA, USA) and were two previously undescribed species (Damm et al. 2009). sequenced directly with the BigDye Terminator v3.1 Cycle Approximately 40 and 90 strains putatively belonging Sequencing kit (Applied Biosystems). Sequencing reactions to the C. graminicola species complex and the RG 9-13 were conducted according to the manufacturer’s instruc- spp., respectively, are preserved at the NIAS Genebank, tions. Extension products were analyzed on an ABI PRISM National Institute of Agrobiological Sciences, Japan. Acces- 3730 DNA Analyzer (Applied Biosystems, Foster City, sions in RG 9-13 spp. are more important economically in CA, USA) according to the manufacturer’s instructions. All Japan because they contain numerous horticultural and food sequences were uploaded to the database, “Microorganism crop pathogens (The Phytopathological Society of Japan & Search System”, NIAS Genebank (http://www.gene.affrc. National Institute of Agrobiological Science 2012). Thus, in go.jp/databases-micro_search_en.php). order to appropriately control anthracnose caused by these For phylogenetic analyses, sequence data of the ITS species, it is important that we re-identify these fungi based region and the GAPDH, CHS-1, HIS3, ACT and TUB2 on the latest methods of molecular phylogenetic analysis gene sequences for the 83 strains examined in this paper, and clarify host ranges and morphological differences as well as 22 strains comprising an additional 18 species among species. We, therefore, re-identified a number of revised by Damm et al. (2009) downloaded from DDBJ/ Japanese strains belonging to RG 9-13 according to the EMBL/GenBank databases, were included as references method described by Damm et al. (2009) to characterize the (Table 2). Sequences for strains of Glomerella lindemuthi- distribution of the species revised by Damm et al. (2009) ana Shear (Colletotrichum lindemuthianum (Sacc. & Mag- and their host plants. The geometry of conidial morphology, nus) Briosi & Cavara) deposited in the database were also especially the curvature of conidia, was also examined to used as an outgroup. Multiple sequence alignments were quantify the morphological characteristics of the re-identi- carried out using the FFT-NS-i strategy of MAFFT version fied species. 6 (Katoh et al. 2002). The alignments of all sequences were further optimized manually, and gaps were deleted. Materials and Methods A phylogenetic tree was constructed from sequences of the six genes combined by maximum likelihood (ML) methods 1. Molecular phylogenetic analyses and re-identifica- using shotgun searches with RAxML version 8 (Stamaka- tion of strains kis 2014). The search was repeated until the maximum Eighty-three strains belonging to RG 9-13 preserved likelihood was identified. Base composition homogeneity at the NIAS Genebank (Table 1) were re-identified based tests were conducted using Kakusan4 (Tanabe 2011). The on phylogenetic analyses using the ITS region of the rRNA model: GTR + Gamma was used in the tree searches. The gene, sequences of the glyceraldehyde 3-phosphate dehy- reliability of the inferred tree was estimated by bootstrap drogenase (GAPDH), chitin synthase 1 (CHS-1), histone3 analysis (Felsenstein 1985) repeated 100 times. Another (HIS3) and actin (ACT) genes, and partial sequences of ML tree was constructed with TUB2 gene partial sequences 352 JARQ 49 (4) 2015 Anthracnose Fungi, Colletotrichum spp. with Curved Conidia in Japan only to estimate the efficacy of using a single gene for the System” on the NIAS Genebank website (http://www. molecular identification of species belonging to RG 9-13. gene.affrc.go.jp/databases-micro_search_en.php). Plants Partial sequences of the TUB2 gene from the seven reported to be susceptible to the fungal strains were deter- strains listed in Table 1 were used in “Standard Nucleotide mined by search of the references listed on the “Detailed BLAST” searches on the NCBI website (http://www.ncbi.